1. Field of the Invention
This invention relates to a reagent and method for demonstrating and determining calcium in a liquid sample and, more particularly, this invention relates to means for stabilizing an aqueous solution of methylthymol blue dye, or similar dye, in a reagent useful in assaying calcium in biologic fluids.
2. Brief Description of the Prior Art
Gindler U.S. Pat. No. 3,754,865 (Aug. 28, 1973), the disclosure of which is hereby incorporated by reference, discloses a reagent and method for demonstrating and determining calcium in biologic fluids. According to the Gindler patent, methylthymol blue, or a similar phenolsulphonephthalein dye, present in aqueous solution in a diagnostic reagent binds with calcium in a liquid sample to produce a colored complex, the concentration of which is directly and linearly related to the concentration of calcium in the sample, and which can be readily determined using standard spectrophotometric techniques.
The Gindler patent further discloses that interference from magnesium present in the sample can be eliminated by the presence of a sequestering agent such as 8-quinolinol (8-hydroxyquinoline) which complexes with magnesium, thus preventing magnesium from complexing with the dye.
The preferred reagent of the Gindler patent also includes a micelle-forming protective colloid (which holds lipids in solution, thus preventing turbidity, and eliminates errors due to the presence of protein), a reducing agent (which stabilizes the color of the calcium dye complex) and a buffering agent (which prevents absorption of light by uncomplexed dye). The reagent is effective in assaying calcium even in samples which contain proteins, phosphates and bilirubin.
Methylthymol blue dye, and similar phenolsulphonephthalein dyes, are known to be chemically unstable in aqueous solution. The Gindler patent discloses that this is not the case with respect to acidic solutions of the dye which, preferably, contain a protective colloid and a magnesium complexing agent. Gindler discloses that such aqueous solutions of methylthymol blue can be stabilized for as long as about one year by maintaining the pH of the solution below about 4 by the addition of a strong acid having a water-soluble calcium salt to the solution in an amount sufficient to achieve the desired pH.
This teaching of the Gindler patent is consistent with the theory that phenolsulphonephthalein dye instability is a result of base-catalyzed hydrolysis of amino groups present in the dye molecule.
Practical experience with a commercial form of the reagent described in the Gindler patent has revealed that, even with the presence of strong acids, methylthymol blue dye is somewhat unstable. Such instability contributes to a rise in optical density of blank solutions containing the reagent, and a drop in linearity of the colorimetric reaction. Specifically, rises in blank optical density to greater than about 1.3, followed by drops in linearity of up to 10%, have been experienced.